Substrate Ligand Research Articles

New Insights into the Structural Features and Functional Relevance of Human Cytochrome P450 2C9. Part II

Part I of this article published in the previous issue of Current Drug Metabolism discussed the substrate specificity, inhibitor selectivity and structure-activity relationship (SAR) of human CYP2C9. The features of CYP2C9 pharmacophore and SAR models have been elaborated. Part II of this article will address the homology models of CYP2C9, data from site-directed mutagenesis studies, and crystal structural features of CYP2C9. The heteroactivation of CYP2C9 and its interactions with other CYPs will also be discussed. A number of ligand-based and homology models of CYP2C9 have been reported and this has provided insights into the binding of ligands to the active site of CYP2C9. Site-directed mutagenesis studies have revealed that a number of residues (e.g. R97, F110, F114, R132, R144, D293, F476 and A477) play an important role in ligand binding and determination of substrate specificity. The resolved crystal structures of CYP2C9 have confirmed the importance of these residues in substrate recognition and ligand orientation. Currently, there are three X-ray structures of the human CYP2C9 in Protein Database (PDB): one ligand-free protein (1OG2), and two in complex with S-warfarin (1OG5) or flurbiprofen (1R9O). The published structures of 1OG2 and 1OG5 differ in comparison with 1R9O in residues 30-53 of N-termini, residues 97-121 of B/C-loops, and residues 196-233 of helix F and F/G-loops. CYP2C9 is a two-domain protein with typical fold characteristics of the CYPs. The B-C loop forms part of the active site and contributes to substrate specificity. In the structures of CYP2C9 without ligand bound or with bound S-warfarin, residues 101-106 in the B-C loop form helix B'. In addition, residues 212-222 in the F-G loop form helices F' and G', which was not observed in rabbit CYP2C5 and bacterial CYPs. In the 1OG2 and 1OG5 structures, the heme is stabilized by hydrogen bonds between the propionates and the side chains of W120, R124, H368 and R433. In addition, R97 forms hydrogen bonds to the propionates as well as the carbonyl oxygen atoms of V113 and P367. CYP2C9 is activated by dapsone and its analogues and R-lansoprazole in a stereo-specific and substrate-dependent manner, probably through binding to the active site and inducing positive cooperativity. Further studies are needed to investigate the molecular determinants for ligand-CYP2C9 interactions.

Structural tightening and interdomain communication in the catalytic cycle of phosphoglycerate kinase.

Changes in amide-NH chemical shift and hydrogen exchange rates as phosphoglycerate kinase progresses through its catalytic cycle have been measured to assess whether they correlate with changes in hydrogen bonding within the protein. Four representative states were compared: the free enzyme, a product complex containing 3-phosphoglyceric acid (3PG), a substrate complex containing ADP and a transition-state analogue (TSA) complex containing a 3PG-AlF(4)(-)-ADP moiety. There are an overall increases in amide protection from hydrogen exchange when the protein binds the substrate and product ligands and an additional increase when the TSA complex is formed. This is consistent with stabilisation of the protein structure by ligand binding. However, there is no correlation between the chemical shift changes and the protection factor changes, indicating that the protection factor changes are not associated with an overall shortening of hydrogen bonds in the protected ground state, but rather can be ascribed to the properties of the high-energy, exchange-competent state. Therefore, an overall structural tightening mechanism is not supported by the data. Instead, we observed that some cooperativity is exhibited in the N-domain, such that within this domain the changes induced upon forming the TSA complex are an intensification of those induced by binding 3PG. Furthermore, chemical shift changes induced by 3PG binding extend through the interdomain region to the C-domain beta-sheet, highlighting a network of hydrogen bonds between the domains that suggests interdomain communication. Interdomain communication is also indicated by amide protection in one domain being significantly altered by binding of substrate to the other, even where no associated change in the structure of the substrate-free domain is indicated by chemical shifts. Hence, the communication between domains is also manifested in the accessibility of higher-energy, exchange-competent states. Overall, the data that are consistent with structural tightening relate to defined regions and are close to the 3PG binding site and in the hinge regions of 3-phosphoglycerate kinase.

Substrates, Inducers, Inhibitors and Structure-Activity Relationships of Human Cytochrome P450 2C9 and Implications in Drug Development

Cytochrome P450 2C9 (CYP2C9) is one of the most abundant CYP enzymes in the human liver. CYP2C9 metabolizes more than 100 therapeutic drugs, including tolbutamide, glyburide, diclofenac, celecoxib, torasemide, phenytoin losartan, and S-warfarin). Some natural and herbal compounds are also metabolized by CYP2C9, probably leading to the formation of toxic metabolites. CYP2C9 also plays a role in the metabolism of several endogenous compounds such as steroids, melatonin, retinoids and arachidonic acid. Many CYP2C9 substrates are weak acids, but CYP2C9 also has the capacity to metabolise neutral, highly lipophilic compounds. A number of ligand-based and homology models of CYP2C9 have been reported and this has provided insights into the binding of ligands to the active site of CYP2C9. Data from the site-directed mutagenesis studies have revealed that a number of residues (e.g. Arg97, Phe110, Val113, Phe114, Arg144, Ser286, Asn289, Asp293 and Phe476) play an important role in ligand binding and determination of substrate specificity. The resolved crystal structures of CYP2C9 have confirmed the importance of these residues in substrate recognition and ligand orientation. CYP2C9 is activated by dapsone and its analogues and R-lansoprazole in a stereo-specific and substrate-dependent manner, probably through binding to the active site and inducing positive cooperativity. CYP2C9 is subject to induction by rifampin, phenobarbital, and dexamethasone, indicating the involvement of pregnane X receptor, constitutive androstane receptor and glucocorticoid receptor in the regulation of CYP2C9. A number of compounds have been found to inhibit CYP2C9 and this may provide an explanation for some clinically important drug interactions. Tienilic acid, suprofen and silybin are mechanism-based inhibitors of CYP2C9. Given the critical role of CYP2C9 in drug metabolism and the presence of polymorphisms, it is important to identify drug candidates as potential substrates, inducer or inhibitors of CYP2C9 in drug development and drug discovery scientists should develop drugs with minimal interactions with this enzyme. Further studies are warranted to explore the molecular determinants for ligand-CYP2C9 binding and the structure-activity relationships.

Effects of Substrate, Protein Environment, and Proximal Ligand Mutation on Compound I and Compound 0 of Chloroperoxidase

This paper investigates the enzyme chloroperoxidase (CPO) by means of hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. The effects of anionic substrate, protein environment, and proximal ligand mutation on the high-valent iron-oxo species, compound I (Cpd I), and the ferric hydroperoxide complex, compound 0 (Cpd 0), are studied. The results indicate that the presence of an anionic substrate (acetate) and the protonation state of one critical residue (Glu104) have a considerable impact on the relative stabilities of Cpd I and Cpd 0. In the absence of the substrate or when the substrate is protonated, Cpd I is considerably more stable, and its formation barrier is smaller than in the case where the substrate is in its anionic state and when Glu104 is deprotonated. This trend, which is shown to be a simple manifestation of the Hammond principle, reproduces the experimental observation that the working pH of the enzyme is acidic. Furthermore, in the absence of substrate (or when it is protonated), the relative Cpd 0/Cpd I energies are found to be a good index of Cpd I stability in heme enzymes and to follow the experimental order: horseradish peroxidase (HRP) > CPO > P450. In silico mutation of the proximal ligand from cysteine to selenocysteine was found to have no effect at all on the properties of Cpd I (e.g., spin density on the chalcogen, Mössbauer parameters, etc.) and its relative stability to Cpd 0 or on the corresponding barrier for formation. This surprising finding shows that the polar CPO pocket applies a leveling effect that stabilizes the anionic forms of the proximal ligands (CysS(-) and CysSe(-)). This in turn means that the Se-Cpd I of the mutant CPO is observable.

Allosteric Communication Occurs via Networks of Tertiary and Quaternary Motions in Proteins

Allosteric proteins bind an effector molecule at one site resulting in a functional change at a second site. We hypothesize that allosteric communication in proteins relies upon networks of quaternary (collective, rigid-body) and tertiary (residue–residue contact) motions. We argue that cyclic topology of these networks is necessary for allosteric communication. An automated algorithm identifies rigid bodies from the displacement between the inactive and the active structures and constructs “quaternary networks” from these rigid bodies and the substrate and effector ligands. We then integrate quaternary networks with a coarse-grained representation of contact rearrangements to form “global communication networks” (GCNs). The GCN reveals allosteric communication among all substrate and effector sites in 15 of 18 multidomain and multimeric proteins, while tertiary and quaternary networks exhibit such communication in only 4 and 3 of these proteins, respectively. Furthermore, in 7 of the 15 proteins connected by the GCN, 50% or more of the substrate-effector paths via the GCN are “interdependent” paths that do not exist via either the tertiary or the quaternary network. Substrate-effector “pathways” typically are not linear but rather consist of polycyclic networks of rigid bodies and clusters of rearranging residue contacts. These results argue for broad applicability of allosteric communication based on structural changes and demonstrate the utility of the GCN. Global communication networks may inform a variety of experiments on allosteric proteins as well as the design of allostery into non-allosteric proteins.

Dynamics of Ligand Binding from 13C NMR Relaxation Dispersion at Natural Abundance

We show that Carr-Purcell-Meiboom-Gill (CPMG) 13Calpha NMR relaxation dispersion measurements are a viable means for profiling mus-ms ligand dynamics involved in receptor binding. Critically, the dispersion is at natural 13C abundance; this matches typical pharmaceutical research settings in which ligand isotope-labeling is often impractical. The dispersion reveals ligand 13Calpha nuclei that experience mus-ms modulation of their chemical shifts due to binding. 13Calpha shifts are dominated by local torsion angles , psi, chi1; hence, these experiments identify flexible torsion angles that may assist complex formation. Since the experiments detect the ligand, they are viable even in the absence of a receptor structure. The mus-ms dynamic information gained helps establish flexibility-activity relationships. We apply these experiments to study the binding of a phospho-peptide substrate ligand to the peptidyl-prolyl isomerase Pin1.

Catalytic Scaffolding Ligands: An Efficient Strategy for Directing Reactions

The design and application of a scaffolding ligand that promotes branch and diastereoselective hydroformylation of terminal olefins as well as the regio- and diastereoselective hydroformylation of disubstituted olefins is reported. It is shown that the ligand covalently and reversibly bonds to the substrate, allowing for directed hydroformylation. As the substrate ligand interaction is dynamic, hydroformylations are catalytic in ligand and do not require any additional synthetic steps to add or remove the directing group. Using a catalytic quantity of a scaffolding ligand (20-25 mol %), excellent regioselectivity for disubstituted olefins (up to 98:2) and high branch selectivity (up to 88:12) for terminal olefins were obtained.

Interaction between Substrate and Oxygen Ligand Responsible for Effective O–O Bond Cleavage in Bovine Cytochrome P450 Steroid 21-Hydroxylase Proved by Raman Spectroscopy

We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17alpha-hydroxyprogesterone. The uncoupled H(2)O(2) formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17alpha-hydroxyprogesterone (15% of NADPH consumed), indicating that 17alpha-hydroxyprogesterone can better facilitate the O-O bond scission. In relation to this, it is noted that the O-O stretching mode (nu(O-O)) of the oxygen complex of P450c21 was sensitive to the substrate; the progesterone- or 17alpha-hydroxyprogesterone-bound enzyme gave single (at 1137 cm(-1)) or split nu(O-O) bands (at 1124 and 1138 cm(-1)), respectively, demonstrating the presence of two forms for the latter. In contrast to nu(O-O), no corresponding difference was observed for the Fe-O(2) stretching mode between two different substrate-bound forms. The Fe-S(Cys) stretching mode in the ferric state was also identical (349 cm(-1)) for each substrate-bound form, suggesting that modulation through the axial thiolate by the substrate is unlikely. Therefore, it is deduced that the hydroxyl group at C-17 of 17alpha-hydroxyprogesterone forms a hydrogen bond with the terminal oxygen atom of the FeOO complex in one form, yielding a lower nu(O-O) frequency with higher reactivity for O-O cleavage, whereas the other form in which the substrate does not provide a hydrogen bond to the oxygen ligand is essentially the same between the two kinds of substrates. In the hydrogen-bonded species, the substrate changes the geometry of the FeOO moiety, thereby performing the hydroxylation reaction more effectively in 17alpha-hydroxyprogesterone than in progesterone.

Dopamine transporter comparative molecular modeling and binding site prediction using the LeuTAa leucine transporter as a template

Pharmacological and behavioral studies indicate that binding of cocaine and the amphetamines by the dopamine transporter (DAT) protein is principally responsible for initiating the euphoria and addiction associated with these drugs. The lack of an X-ray crystal structure for the DAT or any other member of the neurotransmitter:sodium symporter (NSS) family has hindered understanding of psychostimulant recognition at the atomic level; structural information has been obtained largely from mutagenesis and biophysical studies. The recent publication of a crystal structure for the bacterial leucine transporter LeuT(Aa), a distantly related NSS family homolog, provides for the first time a template for three-dimensional comparative modeling of NSS proteins. A novel computational modeling approach using the capabilities of the Molecular Operating Environment program MOE 2005.06 in conjunction with other comparative modeling servers generated the LeuT(Aa)-directed DAT model. Probable dopamine and amphetamine binding sites were identified within the DAT model using multiple docking approaches. Binding sites for the substrate ligands (dopamine and amphetamine) overlapped substantially with the analogous region of the LeuT(Aa) crystal structure for the substrate leucine. The docking predictions implicated DAT side chains known to be critical for high affinity ligand binding and suggest novel mutagenesis targets in elucidating discrete substrate and inhibitor binding sites. The DAT model may guide DAT ligand QSAR studies, and rational design of novel DAT-binding therapeutics.

Methylene substitution at the α–β bridging position within the phosphate chain of dUDP profoundly perturbs ligand accommodation into the dUTPase active site

dUTP pyrophosphatase, a preventive DNA repair enzyme, contributes to maintain the appropriate cellular dUTP/dTTP ratio by catalyzing dUTP hydrolysis. dUTPase is essential for viability in bacteria and eukaryotes alike. Identification of species-specific antagonists of bacterial dUTPases is expected to contribute to the development of novel antimicrobial agents. As a first general step, design of dUTPase inhibitors should be based on modifications of the substrate dUTP phosphate chain, as modifications in either base or sugar moieties strongly impair ligand binding. Based on structural differences between bacterial and human dUTPases, derivatization of dUTP-analogous compounds will be required as a second step to invoke species-specific character. Studies performed with dUTP analogues also offer insights into substrate binding characteristics of this important and structurally peculiar enzyme. In this study, alpha,beta-methylene-dUDP was synthesized and its complex with dUTPase was characterized. Enzymatic phosphorylation of this substrate analogue by pyruvate kinase was not possible in contrast to the successful enzymatic phosphorylation of alpha,beta-imino-dUDP. One explanation for this finding is that the different bond angles and the presence of the methylene group may preclude formation of a catalytically competent complex with the kinase. Crystal structure of E. coli dUTPase:alpha,beta-methylene-dUDP and E. coli dUTPase:dUDP:Mn complexes were determined and analyzed in comparison with previous data. Results show that the "trans" alpha-phosphate conformation of alpha,beta-methylene-dUDP differs from the catalytically competent "gauche" alpha-phosphate conformation of the imino analogue and the oxo substrate, manifested in the shifted position of the alpha-phosphorus by more than 3 A. The three-dimensional structures determined in this work show that the binding of the methylene analogue with the alpha-phosphorus in the "gauche" conformation would result in steric clash of the methylene group with the protein atoms. In addition, the metal ion cofactor was not bound in the crystal of the complex with the methylene analogue while it was clearly visible as coordinated to dUDP, arguing that the altered phosphate chain conformation also perturbs metal ion complexation. Isothermal calorimetry titrations indicate that the binding affinity of alpha,beta-methylene-dUDP toward dUTPase is drastically decreased when compared with that of dUDP. In conclusion, the present data suggest that while alpha,beta-methylene-dUDP seems to be practically nonhydrolyzable, it is not a strong binding inhibitor of dUTPase probably due to the altered binding mode of the phosphate chain. Results indicate that in some cases methylene analogues may not faithfully reflect the competent substrate ligand properties, especially if the methylene hydrogens are in steric conflict with the protein.

Unraveling the three-metal-ion catalytic mechanism of the DNA repair enzyme endonuclease IV

Endonuclease IV belongs to a class of important apurinic/apyrimidinic endonucleases involved in DNA repair. Although a structure-based mechanistic hypothesis has been put forth for this enzyme, the detailed catalytic mechanism has remained unknown. Using thermodynamic integration in the context of ab initio quantum mechanics/molecular mechanics molecular dynamics, we examined certain aspects of the phosphodiester cleavage step in the mechanism. We found the reaction proceeded through a synchronous bimolecular (A(N)D(N)) mechanism with reaction free energy and barrier of -3.5 and 20.6 kcal/mol, in agreement with experimental estimates. In the course of the reaction the trinuclear active site of endonuclease IV underwent dramatic local conformational changes: shifts in the mode of coordination of both substrate and first-shell ligands. This qualitative finding supports the notion that structural rearrangements in the active sites of multinuclear enzymes are integral to biological function.

A Pre-Steady State Analysis of Ligand Binding to Human Glucokinase: Evidence for a Preexisting Equilibrium

Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.

Structural and Spectroscopic Characterization of P450 BM3 Mutants with Unprecedented P450 Heme Iron Ligand Sets: NEW HEME LIGATION STATES INFLUENCE CONFORMATIONAL EQUILIBRIA IN P450 BM3

Two novel P450 heme iron ligand sets were generated by directed mutagenesis of the flavocytochrome P450 BM3 heme domain. The A264H and A264K variants produce Cys-Fe-His and Cys-Fe-Lys axial ligand sets, which were validated structurally and characterized by spectroscopic analysis. EPR and magnetic circular dichroism (MCD) provided fingerprints defining these P450 ligand sets. Near IR MCD spectra identified ferric low spin charge-transfer bands diagnostic of the novel ligands. For the A264K mutant, this is the first report of a Cys-Fe-Lys near-IR MCD band. Crystal structure determination showed that substrate-free A264H and A264K proteins crystallize in distinct conformations, as observed previously in substrate-free and fatty acid-bound wild-type P450 forms, respectively. This, in turn, likely reflects the positioning of the I alpha helix section of the protein that is required for optimal configuration of the ligands to the heme iron. One of the monomers in the asymmetric unit of the A264H crystals was in a novel conformation with a more open substrate access route to the active site. The same species was isolated for the wildtype heme domain and represents a novel conformational state of BM3 (termed SF2). The "locking" of these distinct conformations is evident from the fact that the endogenous ligands cannot be displaced by substrate or exogenous ligands. The consequent reduction of heme domain conformational heterogeneity will be important in attempts to determine atomic structure of the full-length, multidomain flavocytochrome, and thus to understand in atomic detail interactions between its heme and reductase domains.

The Long α-Helix of SecA Is Important for the ATPase Coupling of Translocation

SecA contains two ATPase folds (NBF1 and NBF2) and other interaction/regulatory domains, all of which are connected by a long helical scaffold domain (HSD) running along the molecule. Here we identified a functionally important and spatially adjacent pair of SecA residues, Arg-642 on HSD and Glu-400 on NBF1. A charge-reversing substitution at either position as well as disulfide tethering of these positions inactivated the translocation activity. Interestingly, however, the translocation-inactive SecA variants fully retained the ability to up-regulate the ATPase in response to a preprotein and the SecYEG translocon. The translocation defect was suppressible by second site alterations at the hinge-forming boundary of NBF2 and HSD. Based on these results, we propose that the motor function of SecA is realized by ligand-activated ATPase engine and its HSD-mediated conversion into the mechanical work of preprotein translocation.

The effect of direct steric interaction between substrate substituents and ligand substituents on enantioselectivities in asymmetric addition of diethylzinc to aldehydes catalyzed by sterically congested ferrocenyl aziridino alcohols

The direct strong steric interaction between substrate substituents and ligand substituents was first discovered in asymmetric addition of diethylzinc to aldehydes catalyzed by sterically congested ferrocenyl aziridino alcohol derivatives. In addition, this nonbonded steric repulsion influenced significantly enantioselectivities of this reaction, and even led to inversion of the absolute configuration. This fact was further confirmed by the theoretical calculations and the design of a new chiral ferrocenyl aziridino alcohol ligand. A plausible mechanism for this asymmetric reaction was also proposed.

Palmitoyl carnitine, a lysophospholipase-transacylase inhibitor, prevents Candida adherence in vitro

Candida adherence is poorly understood. The results of this study indicate that interactions of Candida with the lyso-forms of phospholipids may be one important attachment mechanism. C. tropicalis and C. albicans adhered to purified lysophospholipids immobilized on microtiter wells, as well as to a human laryngeal epidermoid carcinoma (HEp-2) cell line. Adherence to both lysophospholipids and HEp-2 cells was significantly reduced by palmitoyl carnitine, a lysophospholipase-transacylase inhibitor. Over time there was a positive correlation between Candida adherence and its transacylase activity. The data suggest that palmitoyl carnitine interferes with Candida adherence to lysophospholipids and the HEp-2 cell line by blocking the interaction between the Candida-associated transacylase enzyme receptor site and its lysophospholipid substrate ligand.

The Dynamics and Mechanics of Endothelial Cell Spreading

Cell adhesion to extracellular matrix is mediated by receptor-ligand interactions. When a cell first contacts a surface, it spreads, exerting traction forces against the surface and forming new bonds as its contact area expands. Here, we examined the changes in shape, actin polymerization, focal adhesion formation, and traction stress generation that accompany spreading of endothelial cells over a period of several hours. Bovine aortic endothelial cells were plated on polyacrylamide gels derivatized with a peptide containing the integrin binding sequence RGD, and changes in shape and traction force generation were measured. Notably, both the rate and extent of spreading increase with the density of substrate ligand. There are two prominent modes of spreading: at higher surface ligand densities cells tend to spread isotropically, whereas at lower densities of ligand the cells tend to spread anisotropically, by extending pseudopodia randomly distributed along the cell membrane. The extension of pseudopodia is followed by periods of growth in the cell body to interconnect these extensions. These cycles occur at very regular intervals and, furthermore, the extent of pseudopodial extension can be diminished by increasing the ligand density. Measurement of the traction forces exerted by the cell reveals that a cell is capable of exerting significant forces before either notable focal adhesion or stress fiber formation. Moreover, the total magnitude of force exerted by the cell is linearly related to the area of the cell during spreading. This study is the first to monitor the dynamic changes in the cell shape, spreading rate, and forces exerted during the early stages (first several hours) of endothelial cell adhesion.

Catalytic Cycling in β-Phosphoglucomutase: A Kinetic and Structural Analysis,

Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine beta-PGM activation by the Mg(2+) cofactor, beta-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 A resolution X-ray crystal structure of the Mg(2+)-beta-PGM complex is examined in the context of previously reported structures of the Mg(2+)-alpha-d-galactose-1-phosphate-beta-PGM, Mg(2+)-phospho-beta-PGM, and Mg(2+)-beta-glucose-6-phosphate-1-phosphorane-beta-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg(2+) in the different complexes shows that a single Mg(2+) coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg(2+) anchoring in beta-PGM is consistent with the relatively loose binding indicated by the large K(m) for Mg(2+) activation (270 +/- 20 microM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analyses of the specificity of beta-PGM toward phosphoryl group donors and the specificity of phospho-beta-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of beta-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of beta-PGM by the substrate beta-G1P accounts for the origin of phospho-beta-PGM in the cell.

Crystallographic and Thermodynamic Analysis of the Binding of S-Octylglutathione to the Tyr 7 to Phe Mutant of Glutathione S-Transferase from Schistosoma japonicum,

Glutathione S-transferases are a family of multifunctional enzymes involved in the metabolism of drugs and xenobiotics. Two tyrosine residues, Tyr 7 and Tyr 111, in the active site of the enzyme play an important role in the binding and catalysis of substrate ligands. The crystal structures of Schistosoma japonicum glutathione S-transferase tyrosine 7 to phenylalanine mutant [SjGST(Y7F)] in complex with the substrate glutathione (GSH) and the competitive inhibitor S-octylglutathione (S-octyl-GSH) have been obtained. These new structural data combined with fluorescence spectroscopy and thermodynamic data, obtained by means of isothermal titration calorimetry, allow for detailed characterization of the ligand-binding process. The binding of S-octyl-GSH to SjGST(Y7F) is enthalpically and entropically driven at temperatures below 30 degrees C. The stoichiometry of the binding is one molecule of S-octyl-GSH per mutant dimer, whereas shorter alkyl derivatives bind with a stoichiometry of two molecules per mutant dimer. The SjGST(Y7F).GSH structure showed no major structural differences compared to the wild-type enzyme. In contrast, the structure of SjGST(Y7F).S-octyl-GSH showed asymmetric binding of S-octyl-GSH. This lack of symmetry is reflected in the lower symmetry space group of the SjGST(Y7F).S-octyl-GSH crystals (P6(3)) compared to that of the SjGST(Y7F).GSH crystals (P6(3)22). Moreover, the binding of S-octyl-GSH to the A subunit is accompanied by conformational changes that may be responsible for the lack of binding to the B subunit.

Engineering hepatocellular morphogenesis and function via ligand-presenting hydrogels with graded mechanical compliance

In order to evaluate the sensitivity of hepatocellular cultures to variations in both substrate stiffness and bioactive ligand presentation, hepatocytes were cultured on differentially compliant polyacrylamide gel discs functionalized with varying amounts of the ECM ligand, fibronectin (FN). Subconfluent cell cultures were established in a multiwell plate format enabling the systematic evaluation of cellular response to both underlying substrate rigidity and substrate ligand concentration. Hepatocellular morphogenesis, regulated by a combination of both ligand density and substrate compliance, resulted in a broad spectrum of patterns of cellular reorganization and assembly ranging from highly two-dimensionally spread cells to highly compact, three-dimensional spheroids. Cell compaction was promoted by increasing levels of substrate mechanical compliance and generally inhibited by increasing concentrations of substrate-bound FN. We identified regimes of substrate compliance in which cells are highly responsive or relatively insensitive to the level of substrate-based ligands. For example, while FN presentation did not have a large impact on cell morphogenesis for cultures on highly compliant gels (G' = 1.9 kPa), hepatocytes on "firm" substrates of intermediate compliance (G' = 5.6 kPa) exhibited approximately a 2-fold increase in cell area between the highest and lowest FN concentrations used in this study. Further, we show that increasing substrate compliance at constant ligand concentration results in increased levels of liver-specific albumin secretion while increasing levels of FN at constant substrate rigidity yield reduced liver-specific functional activity. These substrate-elicited differences in cell function also coincided with analogous changes in the transcript levels of metabolic, growth-related, and liver-specific gene markers. Notably, these results also demonstrated that "firm" gel substrates elicit the most hepatocyte functional sensitivity to substrate-based FN presentation. Overall, our findings indicate that hepatocellular responsiveness to ligand concentration can be acutely regulated by gradation of substrate compliance, suggesting that concerted biochemical and biophysical design strategies may be critical toward the fabrication of hepatospecific biomaterials that effectively support desired levels of liver-specific function.